Pathogens produce an enormous variety of factors that interact with host components to promote infections. Many such factors resemble or mimic host proteins. Proteins involved in these host-pathogen interactions are some of the most rapidly evolving factors in genomes. Yet little is known about the consequences of this rapid evolution on host-pathogen relationships. This research program aims to examine rapid evolution of interacting host and pathogen proteins by focusing on the model poxvirus, vaccinia. Aim 1, which will be completed in the K99 phase of the project, investigates the origins and evolution of a fast evolving poxvirus protein called K3L that mimics the substrate of the anti-viral Protein kinase R (PKR) to disrupt anti-viral activity. Evolutionary analysis will guide the reconstruction of ancestral K3L variants, while cellular assays and experimental viral infections will test the potency of these reconstructed evolutionary steps. Aim 2 investigates the evolution of K3L from smallpox, a devastating human pathogen. Experiments focusing on K3L sensitivity to PKR from rodents will test the hypothesis that smallpox emerged from a specific rodent host. Aim 3 of this program entails experimental evolution of vaccinia virus in different host cell lines. Vaccinia will be repeatedly passaged in cell lines under controlled conditions and monitored for genomic changes and potential adaptations. This aim, along with Aim 2, will be initiated during the K99 phase of the project, and will provide extensive data on evolutionary adaptations between interacting host and pathogen factors. The data generated from these experiments will be the foundation of continuing projects proposed in Aim 3 that will be conducted during the independent phase of the project. All the experimental aims of this program will develop important skills for establishing an independent research laboratory. This investigation of the evolutionary dynamics between interacting host and pathogen factors will provide new insights into the evolutionary strategies of poxviruses, a potentially dangerous class of pathogens poised for natural epidemics and/or use as agents of bio-terrorism. Public Health Relevance: Poxviruses are large DNA viruses that infect animals and humans. Smallpox is a Center for Disease Control high-threat (Category A) agent and monkeypox may be poised for epidemic infections of human populations. This project takes an evolutionary approach to understanding how poxviruses adapt to exploit their hosts, so that these evolutionary strategies might be counteracted for the protection of human health.